Amine functionalized superparamagnetic nanoparticles for the synthesis of bioconjugates and uses therefor

ABSTRACT

Amine functionalized magnetic nanoparticle compositions and processes for synthesizing the same are described. The process consists of obtaining a carboxylated polymer in substantially pure form, which is used to prepare a substantially size homogeneous, polymer coated carboxyl, functionalized magnetic nanoparticle. The carboxyl groups are converted to reactive primary amino groups by the use of a water-soluble carbodiimide followed by reaction of a large excess of a diamine. The amine-terminated nanoparticles are then reacted with bifunctional crosslinking agents and with various biomolecules to make nanoparticles for in vitro assays, cell sorting applications and target specific MR contrast agents.

RELATED APPLICATIONS

[0001] This Application claims priority under 35 USC 119(e) toprovisional patent application U.S. No. 60/345,233 filed Jan. 2, 2002.

TECHNICAL FIELD

[0002] The present invention relates to novel superparamagneticnanoparticle compositions, synthesis and uses thereof.

BACKGROUND

[0003] All patents, patent applications and references cited in thisspecification are incorporated herein by reference.

[0004] Carboxyl bearing polymers have found a wide usage in food, drugs,and industrial applications. A few well known examples include thecarboxymethyl (‘CM’) polysaccharides CM-cellulose, CM-dextran, andCM-arabinogalactan (U.S. Pat. No. 5,981,507). The CM polysaccharides areproduced from a reaction of the polysaccharide with haloacetic acids inbase.

[0005] Carboxyl bearing polymers have been used in the synthesis ofmagnetic nanoparticles. Carboxylated dextrans of two major types havebeen used in the synthesis of superparamagnetic iron oxidenanoparticles. Carboxydextrans have a single terminal carboxyl group oneach dextran molecule obtained by treatment with base (Hasegawa U.S.Pat. No. 4,101,435; Hasegawa U.S. Pat. No. 5,424,419, column 2, line17). Carboxymethylated dextrans have numerous carboxymethyl groupsattached per mole of dextran by reaction of alkyl halogenated acids inbase (Maruno U.S. Pat. No. 5,204,457; Groman WO 00/61191).

[0006] Magnetic nanoparaticles used for the attachment of biomoleculeshave been described by Molday (U.S. Pat. No. 4,452,773). A dextrancoated magnetic nanoparticle is formed and then treated with periodateto produce aldehyde groups. The aldehydes react with amino groups on abiological molecule, to form a Schiff base. The Schiff base maybestabilized by treatment of with a reducing agent like sodiumborohydride. After treatment with a reducing agent a methylene aminolinker connects the biomolecule to the nanoparticle. As shown in FIG.2B, there are no peptidyl bonds in such linkages. A drawback of thismethod is the difficulty controlling the number and position of aminogroups on the biomolecule that are available to react with the reactivealdehyde groups on the nanoparticle.

[0007] Other methods of attaching biomolecules to nanoparticles also usethe reactivity of the aldehyde group. Rembaum and coworkers haveutilized this approach, synthesizing nanoparticles with glutaraldehyde(U.S. Pat. Nos. 4,438,239; 4,369,226).

[0008] The development of amine functionalized crosslinked iron oxidenanoparticle (“amino-CLIO”, FIG. 2) by one of the inventors has provento be an excellent method of synthesizing magnetic particle-biomoleculeconjugates. Amino-CLIO is prepared by first synthesizing a dextrancoated magnetic nanoparticle, followed by crosslinking the dextran withepichlorohydrin. Finally the amine groups are incorporated by reactingthe dextran with ammonia (see Josephson et al, (1999) Bioconjug Chem 10,186-91; Josephson et. al (2001) Angwandte Chemie 40, 3204-3206;).

[0009] Amino-CLIO is an excellent label for the attachment ofbiomolecules, and for the synthesis of magnetic nanoparticle-biomoleculeconjugates, for two reasons. First it provides an amine group forreaction with many bifunctional conjugation reagents that consist ofN-hydroxysuccinimide esters that react first with an amine group andhave a second group that reacts with sulfhydryl groups on a biomolecule.Examples of these bifunctional conjugating reagents are SPDP, SIA, SMCCand MBS. These reagents are available commercially (Pierce Chemical orMolecular Biosciences). Examples of biomolecules that have been attachedto amino-CLIO include peptides (Josephson et al, (1999) Bioconjug Chem10, 186-91), oligonucleotides (Josephson et. al (2001) Angwandte Chemie40, 3204-3206;) and proteins (Hogemann et al. (2000). Bioconjug Chem 11,941-6). Second, amino-CLIO is highly stable due to the fact that thecrosslinking forms a shell of dextran around a core of iron oxide. Thisallows storage of either amino-CLIO or bioconjugates based on amino-CLIOunder a wide range of conditions (temperature, pH, ionic strength). Bycovalently joining polymeric molecules of the coating, crosslinking isassociated with a pronounced increase in the molecular weight of thepolymeric coating.

[0010] This amino-CLIO based chemistry has one major drawback, however,which arises precisely because of the extraordinary stability achievedby using a crosslinked-stablilized dextran on the nanoparticle surface.For human parenteral applications, such as for a magnetic label fortargeted MR contrast agents, the degradation or elimination of theagent, including the coating, is required. However, when the iron oxideof an amino-CLIO based MR contrast is dissolved or biodegraded, thecrosslinked dextran remains as a non-degradable sphere ofpolysaccharide. Similarly, non-degradability occurs with micron-sizedmagnetic microspheres where iron oxide is entrapped in anon-biodegradable polymeric shell (see U.S. Pat. Nos. 4,654,267;5,512,439).

[0011] CM-polymers can also function as starting materials for thesynthesis of drugs conjugates or for the attachment of variousbiological molecules. As drug conjugates, CM-arabinogalactan, CM-dextranand polyvinyl alcohol were used as carriers for nucleotide analogues(U.S. Pat. No. 5,981,507). The carboxyl groups were converted to primaryamino groups by reaction with diamines. Biological molecules like araAMPwere then attached to the primary amine of the aminated arabinogalactan,see Josephson, et al. (1996) Antivir Ther 1, 147-56 and U.S. Pat. No.5,478,576.

[0012] In these examples, the CM-polymers, such as CM-arabinogalactan,exist as macromolecules in solution, which allows conditions to beemployed that insure the nearly quantitative conversion of carboxylgroups to amino groups. The absence of protected carboxyl groups allowsessentially all carboxyl groups to be chemically reactive.

[0013] There is a need for a improved magnetic nanoparticles to whichbiomolecules can be attached for use in cell sorting applications, invitro assays, and which can be used as an intravenously administerable,MR contrast agents. The ideal nanoparticle must have a surface chemistryamenable to the efficient attachment of biomolecules with retention oftheir biological activity. It must be highly stable in vitro, bothbefore and after the attachment of biomolecules. Yet it must be labileor degradable in vivo, with the utilization or elimination of all of itscomponents.

SUMMARY

[0014] The present invention relates to aminofunctionalziednanoparticles. In one embodiment, the nanoparticles have a magnetic corehaving one or more magnetic metal oxide crystals, and a noncrosslinkedpolymer coating associated with the core. The polymer coating has aplurality of carboxyl groups and plurality of reactive primary aminogroups. A portion of the carboxyl groups are associated with thecrystals. In one embodiment, a portion of the amino groups areassociated with polymer through a peptidyl linkage of the formula:

—O—(CH₂)_(m)—CONH—[X]

[0015] wherein X is —(CH₂)_(n)NH₂, —(CH₂)_(o)CH NHCOO—,—(CH₂)₃NH(CH₂)₄NH₂ or —(CH₂)₃NH(CH₂)₄NH(CH₂)₃ NH₂—

[0016] wherein m=1, 2, or 3; n=2, 3, 6, and o=3 or 4.

[0017] In one embodiment, the magnetic core has one or moresuperparamagnetic iron oxide crystals. The superparamagnetic core has adiameter between about 1 nm and about 25 nm, preferably between about 3nm and about 10 nm, and more preferably about 5 nm.

[0018] The nanoparticle (core and polymer) has diameter between about 15nm and 100 nm, preferably between about 20 nm and about 100 nm.

[0019] The polymer coating may be made from natural polymers, orsynthetic polymers, or derivatives or each. Nonlimiting examples includepolyvinyl alcohol and carboxymethyldextran In one aspect of theinvention, the nanoparticle can be conjugated to a biomolecule.

[0020] In another aspect of the invention, there is provided ananoparticle biomolecule conjugate. The nanoparticle portion includes amagnetic core having one or more magnetic metal oxide crystals, anoncrosslinked polymer coating associated with the core. As in theprevious emboidiment, the the polymer coating has a plurality ofcarboxyl groups and plurality of reactive primary amino groups. Aportion of the carboxyl groups are associated with the crystals, In oneembodiment, the amino groups are associated with polymer through apeptidyl linkage of the formula:

—O—(CH₂)_(m)—CONH—[X]

[0021] wherein X is —(CH₂)_(n)NH₂, —(CH₂)_(o)CH NHCOO—,—(CH₂)₃NH(CH₂)₄NH₂ or —(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂—

[0022] wherein m=1, 2, or 3; n=2, 3, 6, and o=3 or 4; and

[0023] whereby a a biomolecule is linked to the nanoparticle through theamino group.

[0024] In another aspect of the invention, there is provided a processfor synthesizing an amine functionalized magnetic metal oxidenanoparticle via the steps of (i) obtaining a polymer having a pluralityof carboxyl groups attached thereto, (ii) contacting the polymer withmagnetic metal oxide to produce a coated magnetic metal oxide wherein aportion of said carboxyl groups are associated with the metal oxide; and(iii) reacting the coated magnetic metal oxide of step (ii) with adiamine. In one embodiement the diamine is ethylene diamine; in anotherembodiment the diamine is hexane diamine.

[0025] In another aspect of the invention, the contacting step in thesynthesis further includes the steps of (a) providing a solution ofsoluble iron salts; (b) converting the iron salts into iron oxidecrystals; and (c) removing unassociated polymer. In yet another aspectof the invention, the converting process includes the further steps of(a) heating the iron salts to a temperature between about 4° C. andabout 20° C.; and adding an amount of base to raise the pH to about 8 orhigher to form iron oxides. In another aspect, the process includes thestep of heating the iron oxides to a temperature about 60° C. or morefor at least 30 minutes.

[0026] Another aspect of the invention provides a pharmaceuticalcomposition of the nanoparticle biomolecule conjugates and apharmaceutically acceptable carrier. This composition is particularlyuseful in targeted MR imaging applications.

BRIEF DESCRIPTION OF THE FIGURES

[0027] In all figures, the large sphere represents the core magneticmaterial.

[0028]FIG. 1 shows a reaction scheme for synthesis of aminofunctionalized magnetic nanoparticles. First, a polymer 1 havingmultiple carboxyl groups is obtained. Next, the polymer is used tosynthesize a coated nanoparticle. The core magnetic material is shown asa dark sphere 2. Carboxyl groups face (i) both the magnetic core whichare blocked (arrow 3 b) and (ii) the solvent (arrow 3 a) and areavailable for further reaction. Third, the free carboxyl groups arereacted, here with carbodiimide, to produce the amine functionalizednanoparticle 4.

[0029] FIGS. 2A-C are comparisons of the prior art nanoparticles (FIGS.2A and 2B) with the nanoparticles of the invention (2C).

[0030] In FIG. 2A, the prior art nanoparticles use crosslinked dextranand ammonia. This produces a functionalized particle in which everynitrogen is a primary amine. The polymer is associated with the magneticcore (large sphere) via hydroxyl groups.

[0031] In FIG. 2B, the prior art nanoparticles use non-crosslinkeddextran which has been reacted with periodate to form aldehydes,followed with EDA and a reducing agent. There are two nitrogen atoms forevery primary amine: a terminal amino group (1) linked to the polymerthrough methyl amine groups (the dashed box, showing second amine 2).There are no peptidyl linkages. The polymer is associated with themagnetic core (large sphere) via hydroxyl groups.

[0032]FIG. 2C shows the nanoparticles of the present invention. Thepolymer is noncrosslinked, and the amino group is linked to the polymervia a peptide bond. There are two nitrogen atoms for each primary amine:terminal amine 1 and at peptidyl linkage 2. The noncrosslinked polymeris associated with the magnetic core (large sphere) via carboxyl groups.

DETAILED DESCRIPTION

[0033] The present invention meets the aforementioned requirements byproviding novel compositions of amino functionalized nanoparticles, andtheir methods of synthesis. These novel amino functionalizednanoparticles can serve further as magnetic nanoparticles for thedevelopment biomolecule-magnetic nanoparticle conjugates useful in avariety of in vitro, and in vivo applications.

[0034] Definitions

[0035] A nanoparticle as described and claimed herein is a material witha “core” of magnetic material associated with a polyfunctionalnoncrosslinked polymer. The polymer coating displays a plurality ofcarboxyl groups and reactive primary amino groups; a portion of thecarboxyl groups are associated with the magnetic core; the reactiveprimary amino groups are available for subsequent covalent reactions,e.g., for the attachment of biomolecules. The nanoparticles have anoverall size less than about 100 nm, before conjugation to biomolecules.The overall size of the nanoparticles is about 15 to 100 nm, preferablyabout 20 to 100 nm, more preferably about 40 to 60 nm; about 50 nm isthe most preferred. The polymeric coating can be about 5 to 20 nm thickor more. Size can be determined by laser light scattering by atomicforce microscopy or other suitable techniques.

[0036] The nanoparticle core can be monodisperse (a single crystal of amagnetic material, e.g., metal oxide, such as superparamagnetic ironoxide, per particle) or polydisperse (a plurality of crystals, e.g., 2,3, or 4, per particle). The metal oxides are crystals of about 1-25 nm,preferably about 3-10 nm, and most preferably about 5 nm in diameter.The magnetic metal oxide can also comprise cobalt, magnesium, zinc, ormixtures of these metals with iron. The term “magnetic” as used in thisspecification and the accompanying claims means materials of highpositive magnetic susceptibility.

[0037] In a preferred embodiment, a superparamagnetic form of iron oxideis used. Superparamagnetic iron oxide is one of the highly magneticforms (magnetite, non-stoichiometric magnetite, gamma-ferric oxide) thathas a magnetic moment of greater than about 30 EMU/gm Fe at 0.5 Teslaand about 300 K. When magnetic moment is measured over a range of fieldstrengths, it shows magnetic saturation at high fields and lacksmagnetic remanence when the field is removed.

[0038] The “polymer coating” is a natural or synthetic polymerassociated with the magnetic core that functions to keep the metaloxides dispersed from each other. In one embodiment the polymer“coating” is not a continuous film around the magnetic metal oxide, butis a “mesh” or “cloud” of extended polymer chains attached to andsurrounding the metal oxide. The polymer coating may be a naturalpolymer, a synthetic polymer. The polymer maybe linear, or moderately orhighly branched. In one embodiment, the polymer coating can be carboxydendrimers, commercially available from Sigma-Aldrich, which are highlybranched polycarboxyl polymers. Other embodiments of the polymer coatingare described in detail below.

[0039] A natural polymer is obtained when a pure polymer, such as apolysaccharide, is synthesized by a microorganism, plant or animal andextracted in substantially pure form. Non limiting examples of naturalpolymers include polysaccharides, such as dextran. A synthetic polymeris obtained from nonbiological syntheses, by using standard polymerchemistry techniques known to those in the art to react monomers intopolymers. The polymers may be homopolymers, i.e, synthesized from asingle type of monomer, or co-polymers, i.e., synthesized from two ormore types of monomers. Non-limiting examples of synthetic polymersinclude polymethylmethacrylate polymers and polyvinyl alcohol polymers.

[0040] A crosslinked polymer is one in which functional groups on apolymer chain and/or branches has reacted with functional groups onanother polymer to form polymer networks. Crosslinked polymers arecharacterized herein as being heat stable and resistant to breakdown inbiological systems. A crosslinked polymer has a molecular weightsignificantly higher than the original starting polymer.

[0041] A non-crosslinked polymer is described and claimed herein as apolymer in which few or no individual polymer chains have reacted withthe functional groups of another polymer chain to form theinterconnected polymer networks. A non-crosslinked polymer that has beenfunctionalized is reasonably size homogeneous as compared to thestarting polymer, i.e., the polymer before and after incorporation ofamine and carboxyl functional groups have similar molecular weights andmolecular weight distributions. A small increase in molecular weight ofthe polymer is seen generally as a result of the incorporation offunctional groups onto the polymer and the few crosslinks that may occuron a statistical basis.

[0042] A polycarboxyl (or carboxyl) polymer is a polymer with more thanone carboxyl group per polymer.

[0043] A polyfunctionalpolymer is one with different functional groups,such as amino and carboxyl groups attached to a polymer.

[0044] Description

[0045] We have surprisingly discovered that amino functionalizednanparticles can be synthesized using non crosslinked, carboxylatedpolymers, and that these polymers permit the addition of reactiveprimary amine groups to the polymer. These reactive primary amines areattached to the polymer via peptidyl linkages.

[0046] When the noncrosslinked carboxylated polymers are used in thesynthesis of the nanoparticles, the resulting polymer coatednanoparticle has two classes of carboxyl groups with very differentchemical reactivities. Some carboxyl groups are shielded from furtherchemical reaction by forming a strong bond between the polymer and thesurface of the iron oxide. Some surface carboxyl groups, facing the bulksolvent, can be activated with carbodiimide and converted to reactiveprimary amino groups (FIG. 1). These reactive primary amines can bereacted with bifunctional conjugating agents and then with biomoleculesto form targeted MR contrast agents, or probes for used in biosensors.

[0047] As shown in FIG. 1, the process for synthesizing magneticnanoparticies involves three general steps: (i) obtaining apolycarboxylated polymer; (ii) synthesizing a polycarboxylated coatedmagnetic nanoparticle; and (iii) converting the surface carboxyl groupsto reactive primary amine groups while the remaining carboxyl groupsbind the polymer to the nanoparticle (FIG. 1).

[0048] As a result of this procedure, the magnetic nanoparticles of theinvention have polyfunctional polymeric coating, i.e. one that containsboth amino and carboxyl groups. Since the carboxylated polymer used tosynthesize the magnetic nanoparticle in step (ii) possesses only asingle class of carboxyl groups, it is highly surprising that theresulting polymer coated magnetic nanoparticle consists of two distincttypes of carboxyl groups. One type is bound to the surface of the ironoxide (blocked from further chemical reaction). A second type is exposedto the bulk solvent and available for conversion to reactive primaryamino groups, see FIG. 1. Moreover, the chemistry used to incorporatethe functional groups results in a polymer coating that isnoncrosslinked (i.e, uncrosslinked or minimnally crosslinked).

[0049] The presence of both reactive primary amino and carboxyl groupson the polymeric coating of the magnetic nanoparticles is one of thedistinctive features of the invention. Polymers containing both reactiveprimary amino groups and carboxyl groups are difficult to developbecause of the propensity for self-reaction. In solution at neutral pH,the positively charged carboxyl group and negatively charged amino groupform non-covalent electrostatic bonds. When covalent modification ofamino and carboxyl group containing polymers is attempted, activation ofthe carboxyl groups with a carbodiimide results in the formation ofpeptidyl bonds by reaction amino groups on the same molecule. Thisresults in intrachain crosslinking when the reactive amino groups are onthe same polymer, or interchain crosslinking when the amino group is ona second polymer molecule. Such interchain cross-linking can be inducedin proteins, which present a mixture of amino and carboxyl groups on thesurface, by activating agents like carbodiimide and is used to makeaggregates of soluble proteins. For this reason the synthesis ofpolymers such as peptides containing amino and carboxyl groups requiresuse of protecting groups followed by deprotection. The synthesis usedhere eliminates the protection-deprotection reaction steps.

[0050] Another feature of the amine functionalized nanoparticles of theinvention is that they can be readily degraded to yield their metalsalts and polymer coating. In one embodiment, the degradation yieldsiron salts and the polyfunctional polymeric coating. In vivo, thisresults in the utilization of iron oxide, by incorporation of iron intored blood cells, and by the excretion and/or degradation of thepolycarboxylated polymer. In vitro, the conditions of biodegradation canbe simulated by exposing nanoparticles to mildly acidic pH (3-6) in thepresence of a metal chelator, e.g. citrate or EDTA. This yields ferricion chelates and soluble polyfunctional polymers. The molecular weightof the polyfunctional polymers, bearing amino and carboxyl groups, willbe slightly larger than the polycarboxylated polymers used to synthesizethe nanoparticles due the addition of reactive primary amino groups.

[0051] The amine functionalized nanoparticles of the invention aresynthesized by activation of free carboxyl groups of the nanoparticlewith a water soluble carbodiimide, followed by reaction with a largeexcess of a diamine. The nature of the diamine provides a linker arm ofvarying lengths and chemistries for the attachment of biomolecules.Nonlimiting examples of diamines include ethylenediamine (EDA),propyldiamine, spermidine, spermine, hexanediamine, and diamine aminoacids, such as lysine or omithine.

[0052] Unlike the synthesis for other amine functionalized particles,ammonia is not used to make the amine functionalized nanoparticles ofthe invention. For example, in the synthesis of amino-CLIO, dextrancoated magnetic nanoparticles are reacted with epichlorohydrin, followedby reaction with ammonia. This reaction produces a dextran crosslinked,amine functionalized nanoparticle bearing primary amino groups(H₂N—CH₂—CHOH—CH₂—O-Polymer), as shown in FIG. 2A. If the carbodiimideactivated carboxylated nanoparticles of this invention were reacted withammonia, an amide would be obtained (H₂N—CO—CH₂—O-Polymer). The nitrogenatoms of amides are far less reactive than primary amino groups and notsatisfactory for reaction with the bifunctional conjugating reagentsused to attach biomolecules.

[0053] The reaction with diamine is performed under conditions thatprevent crosslinking between nanoparticles. This is accomplished byusing a large excess of diamine. In general the moles of diamine usedwill exceed the number of carboxyl groups present by a factor of atleast 10. Diamines are cheap and can be used in very large excess.Unreacted diamine (MW<2 kDa) can be separated from amino functionalizednanoparticle (MW>500 kDa) by ultrafiltration. Alternatives toultrafiltration for the removal of unreacted diamine include gelpermeation chromatography, dialysis, and precipitation andresolubilization of the nanoparticle.

[0054] When the carbodiimide activated carboxylated nanoparticles of theinvention are reacted with a large excess of diamine, one of thenitrogen atoms reacts with the carboxyl group to provide a peptide bond,while a second exists as a primary amine suitable for further chemistry.Hence the amino functionalized nanoparticles of the invention have acharacteristic general structure that includes a peptidyl bond and aprimary amino group, see FIGS. 1 and 2C. This characteristic structureis not found with amino functionalized amino-CLIO nanoparticle.Similarly, a peptide bond is not obtained when dextran coated magneticiron oxides are activated by treatment with periodate, followed byreaction with a primary amine and treatment with a reducing agent. Inthis case a methyl amine linkage is obtained (FIG. 2B).

[0055] The presence of primary amino groups on the magneticnanoparticles can be readily ascertained by reaction with amine specificreagents such as TNBS or ninhydrin or SPDP with the in tact magneticnanoparticle. Since the carboxyl groups are protected by the metaloxide, they can be most easily analyzed after digestion of the metaloxide core and isolation of the polymeric coating. Digestion of metaloxide core can be accomplished by treatment with acid and chelator.Typically a pH below 5, or between 2 and 5, is sufficient. Chelatorslike citrate or EDTA enhance the solubility of iron and are added anamount sufficient to bind all metal ions. After digestion, the metal isremoved by passage over a cation exchange column or metal removingchelating column such as Chelex. The polymer is then analyzed by IR andshows characteristic peaks from carboxyl groups. Polymers with carboxylgroups have characteristic absorption frequencies from the carbonylgroup (C═O) of the carboxyl (1780 to 1710 cm−1, strong) and the hydroxylgroup (3000 to 2500 cm−1, broad, variable).

[0056] The polymer obtained after digestion and removal of iron can alsobe characterized by size and compared with the polymer used in thenanoparticle synthesis. The polymer will be slightly larger than thestarting polymer due to addition of amino groups.

[0057] A second property of the amine functionalized polymers of theinvention is presence of at least two nitrogen atoms for each primaryamine due to characteristic general structure (H₂N—X—NH—CO—), see alsoFIG. 2C. X can be any structure connecting the two primary amines of thediamine. Non limiting examples include hexamine diamine, ethylenediamine, spermidine or spermine, and amino acids like omithine orlysine, which are of interest because of their negatively chargedcarboxyl group. The total number of nitrogen groups attached to thepurified polymer can be obtained by submitting the purified polymer toelemental analysis of nitrogen, i.e., determination of the content ofall nitrogen atoms. The number of reactive primary amino groups can bedetermined by the TNBS method. A property of the amine functionalizedpolymers of the invention is the amount of total nitrogen will exceedthe amount of nitrogen present as a primary amine. For example, whenethylene diamine (EDA) has been used, the total nitrogen content will betwice the nitrogen content obtained with methods determining the amountof primary amine.

[0058] Properties and sources of polycarboxylated polymers for thesynthesis of coated nanoparticle. The polycarboxylated polymers can beobtained by a variety of routes and have a variety of compositions. Theymay be man made or naturally occurring and may be highly branched orlinear.

[0059] The polycarboxylated polymers have a molecular weight betweenabout 5 and 200 kDa, more preferably between 5 and 50 kDa. Smallerpolymers lack a sufficient number of carboxyl groups to both stronglybind to the iron oxide and have the numerous free carboxyl groupsavailable for conversion to amino groups (FIG. 1). The polymers mustcontain more than about five moles of carboxyl group per mole ofpolymer. The number of carboxyl groups can be determined by titration.The polycarboxylated polymers should have a high water solubility over awide range of pH's to be employed in the synthesis of water solublepolymer coated magnetic nanoparticles.

[0060] To be used in the synthesis of polymer coated magneticnanoparticles, unreacted polymer must be separated from polymer coatednanoparticle. To do this easily and efficiently, it is preferred thatthe polymer maintain a homogenous size distribution, which can bedetermined by the usual methods of polymer chemistry including lightscattering, gel permeation chromatography. For example, unreactedcarboxymethylated dextran (MW=20 kDa) can be readily separated from thecoated nanoparticle (MW>500 kDa) by ultrafiltration using a membranewith a cutoff of 100 kDa (Groman WO 00/61191). Polymers larger thanabout 200 kDa can used but they are more difficult and more expensive toseparate from the polymer coated nanoparticles as the larger molecularweight polymers and the polymer coated nanoparticles pass through thesame membrane.

[0061] A preferred method of synthesizing polycarboxylated polymers isthe reaction of a water soluble polymer containing multiple amino orhydroxyl groups with an alkyl halogenated acid in aqueous strong base.This method has several advantages. First, the size and sizedistribution of the polymer obtained will be determined the size of thestarting polymer. Hence by selecting a size homogeneous polymer, thesize homogenity of the carboxylated polymer is achieved. The size of thepolycarboxylated polymer will be slightly larger than the startingpolymer due to the addition of carboxyl groups. For details see examples1, 4 and 6, of Josephson et al WO97/21452. Second, polymers with varyingnumber of carboxyl groups on them can be synthesized by varying theamount of halogenated acid, to determine the optimum level ofcarboxylation required for the synthesis of polymer coated magneticnanoparticle, see Groman WO 00/61191. An alternative method is thedirect synthesis of polycarboxylic functional polymers, such aspolymethacrylic acid based polymers.

[0062] Naturally occurring hydroxylated polymers that can be used in thesynthesis of polycarboxylated polymers include polysaccharides likedextran, starch or cellulose. Polyvinyl alcohol is a synthetichydroxylated polymer that can replace naturally occurringpolysaccharides. These hydroxyl group-bearing polymers are reacted withhalogenated acids like bromoacetic acid, chloroacetic acid,bromohexanoic acid and chlorohexanoic acid in the present of strongbase, typically 1-8 M NaOH. The polycarboxylated polymer is thenpurified by ultrafiltration or by precipitation. Alternativelyanhydrides like succinic anhydride can be used for carboxylationpolyhydroxylated polymers, but these result in ester linkages which canundergo slow hydrolysis.

[0063] Reaction of positively charged polymers like polylysine or polyvinyl amine with an anhydride (succininc anhydride, maleic anhydride,DTPA anhydride) provide another method of synthesizing carboxylatedpolymers.

[0064] Hydrolysis of an anhydride containing polymer, such aspolytheyelene-alt-maleic anhydride (Sigma) is another method that canyield a suitable carboxylated polymer.

[0065] Carboxyl group bearing polyamino acids can also be employed aspolycarboxylated polymers., e.g. polyaspartate or polyglutamate.Carboxylated dendrimers are available commercially and are highlybranched synthetic polymers and can be used in the synthesis ofnanoparticles.

[0066] Synthesis of carboxyl terminated, polymer coated nanoparticles.

[0067] Carboxy terminated nanoparticles can be synthesized by mixing thecarboxyl terminated polymer with ferrous and ferric salts. Metals otherthan iron can be used in the synthesis of magnetic metal oxides. Forexample, zinc, manganese or cobalt can partially or completely replacethe ferrous ion during the synthesis of magnetic metal oxides.

[0068] The mixture is enclosed in a jacked reactor for temperaturecontrol and stirred. It is covered to control access of oxygen. Themixture is then brought to controlled temperature between 4 and 20 C.,and a base, such as ammonia, is added. Base is added in a highlycontrolled fashion, either by pumping or by drop wise addition.Sufficient base is added to bring the pH to higher than pH 8, whichcauses the formation of iron oxides. The resulting gel or colloid maybeheated, to induce formation of the highly magnetic iron oxide. Thetemperature of the mixture is heated to above 60 C. for more than 30minutes. Finally, the colloid is allowed to cool and unreacted polymerremoved from the polymer coated nanoparticle. The preferred techniquefor removal is ultrafiltration, using a membrane that has a cutoff thatpermits the carboxylated polymer to pass through, while the largercoated nanoparticle is retained. Alternatives to ultrafiltration are gelfiltration and magnetic separation. Citrate maybe added as stabilizerbut it must be removed by ultrafiltration before use of carbodiimidebecause of its carboxyl groups. Details methods for the synthesis ofpolycarboxylated polymer coated nanoparticles can be found in Groman andManro

[0069] Synthesis of amine terminated, polymer coated nanoparticles.

[0070] The carboxyl groups of the carboxyl terminated nanoparticle arethen activated by the use of a water soluble carbodiimide. Typicallythis is done in a non-amine containing buffer between pH 4.5 and 7. Theactivation with 0.1 M TEMED, pH 4.8 has been found to satisfactory.Activation is typically done at 20-40 C. EDA) is added in vast excess toprevent the formation of crosslinks between the carboxyl groups. Excessdiamine can be separated from the aminated, polymer coated nanoparticleusing ultrafiltration. The use of carbodiimide results in the formationof a peptide bond between the diamine linker and polymeric nanoparticlecoating. The number of primary amines on the particle can be monitoredby reaction with trinitrobenze. A variety of diamines can be used suchas hexamine diamine, ethylene diamine, spermidine or spermine. Aminoacids like omithine or lysine are diamines of interest because of theirnegatively charged carboxyl group.

[0071] The aminofunctionalized nanoparticles can be used as attachmentsubstrates to form a variety of nanoparticle conjugates for in vivo orin vitro applications. Example applications include cell sorting, invitro assays and in vivo applications such as magnetic resonanceimaging. A non limiting exemplary conjugate is a biomoleculenanoparticle conjugate.

[0072] The amino terminated nanoparticle is then reacted with abifunctional conjugation reagent designed to react with amino groups.Preferred conjugation reagents are NHS esters, which react with theamine group of the nanoparticle, and which have second moiety that canreact with the sulfhydryl group of the biomolecule. Such crosslinkingagents include, for example, SPDP, long chain-SPDP, SIA, MBS, SMCC, andothers that are well known in the art and are available from PieceChemical Company. Detailed procedures for their are available from thePiece Chemical web site, see http://www.piercenet.com/ and the attachedpdf files downloaded from that site.

[0073] The activated biomolecule, preferrably with a single sulfhydrylgroup distal from the site of bioactivity, is allowed to react with theactivated nanoparticle. Separation of unreacted biomolecule from thebiomolecule-nanoparticle conjugates can be accomplished by gelfiltration, ultrafiltration, dialysis or magnetic separation methods.Examples of thiolated biomolecules that have been attached to SPDPactivated crosslinked magnetic nanoparpticles include transferrin,(Hogemann, (2000) Bioconjug Chem 11, 941-6), tat peptides (Josephson,(1999) Bioconjug Chem 10, 186-91; Zhao (2002) Bioconjug Chem 13, 840-4),oligonucleotides (Josephson (2001) Agnew Chem Int Ed 40, 3204-3206;Perez, (2002) J Am Chem Soc 124, 2856-7), antibodies (Kang,. (2002)Bioconjug Chem 13, 122-7) and proteins (Perez, Nature Biotechnol 20,816-20). For peptides (1-2 kDa), 5-25 peptides can be attached per 2000Fe atoms. For proteins, such as transferrin or antibodies (50-200 kDa)1-4 biomolecules can be attached per 2000 Fe atoms.

[0074] Uses of the biomolecule-nanoparticle conjugates

[0075] The magnetic nanoparticles of the invention have various useswith in vitro ligand binding assays. The nanoparticles can be used inmagnetic detection based assays (see Simmonds U.S. Pat. Nos. 6,046,585and 6,275,031, Rohr U.S. Pat. No. 5,445,970; Ebersole, U.S. Pat. No.4,219,335, Chemla, et. al. (2000) Ultrasensitive magnetic biosensor forhomogeneous immunoassay. Proc Natl Acad Sci USA 97, 14268-72.). They canalso be used in magnetic resonance based ligand binding assays such asJosephson U.S. Pat. No. 5,164,297 and Perez et al Nature Biotechnol.2002 August;20(8):816-20.

[0076] The magnetic nanoparticles of the invention of the invention arealso suitable for cell sorting applications. Magnetic nanoparticles weredescribed by Molday U.S. Pat. No. 4,452,773 and commercially available(Miltenyi Biotech, Auburn Calif., and Molecular Probes, Eugene Oreg.).

[0077] Finally the magnetic nanoparticles of the invention can be usedas for targeted MR imaging applications.

[0078] For in vivo uses, the biomolecule-nanoparticle conjugates areformulated and sterilized according to the published methods forsterilizing parenterally administered MRI contrast agents. Forparenteral applications, sterilization can be achieved by filtering thecolloid through a 220 nm filter (filter sterilization) or by heatsterilization (terminal sterilization). Depending on the method ofsterilization various excipients, such as monosaccharides,polysaccharides, salts, can be added to stabilize the colloid duringheat stress or storage. Excipients can also serve to bring the ionicstrength and pH of the preparation into the physiological range. SeeJosephson U.S. Pat. No. 5,160,726, Groman U.S. Pat. No. 5,248,492.

EXAMPLES Example 1

[0079] Synthesis of a carboxymethylated polyhydroxylated polymer

[0080] Carboxymethylated polymers are prepared by reaction of a haloacetic acid with a polymer in strong base, usually NaOH. The polymershould be of sufficient molecular weight to allow separation fromunreacted haloacetic acid from the carboxymethylated polymer. Thepolymer is preferably between 5 kDa and 100 kDa. The separation can beaccomplished by dialysis, ultrafiltration or precipitation. The polymeris then dried by lyophilization, vacuum drying or spray drying. Thepolymer should be of sufficient molecular weight to allow separation ofdextran from dextran coated iron oxide. For example, when thenanoparticles have molecular weights of greater than 500 kDa, and thepolymer is preferably less than 100 kDa, this separation can beaccomplished by ultrafiltration.

[0081] (Poly)vinyl alcohol (100 g, MW=15 kDa) was dissolved in 1000 mlof hot water. After reaching room temperature, 600 ml of 8M NaOHsolution was added to it with stirring and again equilibrates to roomtemperature. 100 g of bromo-acetic acid was then added and mixturestirred for two hours. The polymer was neutralized with 6M HCL It wasthen dried under vacuum overnight at room temperature. It is denotedCM-PVA.

Example 2

[0082] Synthesis of carboxymethylated polymer coated nanoparticle

[0083] One hundred milliliters of a solution of 12 mmoles of ferricchloride (hexahydrate) and 6 grams of of CM-PVA. was prepared. Thesolution was filtered and cooled to 2-4 C. To the mixture was added 6mmoles of ferrous chloride (tetrahydrate) dissolved in 5 mL of water.While being stirred rapidly, 4.5 mLs of 28-30% ammonium hydroxide (2-4C.) was added dropwise. The mixture was then heated to between 70 and 90C. and maintained at the higher temperature for 2 hours. UnreactedCM-PVA was removed by ultrafiltration using a 100 kDa cutoff membrane.The colloid had a size of 54 nm by light scattering and an R2 of 60 mM-1sec−1. The procedure was repeated using 3 g CM-PVA to give a colloidwith 65 nm and an R2 of 160 mM−1 sec−1.

Example 3

[0084] Conversion of carboxyl groups on the magnetic nanoparticle toamino groups

[0085] To 10 mLs of 10 mg Fe/mL of the carboxylated polymer coatednanoparticle in 0.1 M TEMED buffer, pH 4.8, was added 0.2 g of1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride at roomtemperature. After 15 mintues, 0.5 mL 1,2 ethylene diamine was added.After 24 hours the mixture was put in dialysis bag and dialyzed untilthe dialysate was free of amine by the TNBS assay.

Example 4

[0086] Reaction of amino-functionalized magnetic nanoparticles with abiomolecule

[0087] Nanoparticles were reacted with N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). To 1 mL of amino functionalizedmagnetic nanoparticle (10 mg Fe) was added 1 mL of 0.1 M phosphatebuffer, pH 7.4, and 2 mL of 25 mM SPDP in DMSO (50 umoles SPDP). Themixture was allowed to stand for 60 min at room temperature. Lowmolecular impurities were removed by PD-10 columns (Sigma Chemical, St.Louis, Mo.) equilibrated with 0.01 MTris and 0.02 Mcitrate, pH 7.4buffer. The number of amine groups can be obtained for the amount of 2PTreleased assayed by addition of dithiothreitol. (Zhao, (2002) BioconjugChem 13, 840-4).

[0088] It will be apparent to those skilled in the art that otherchanges and modifications may be made in the above-described compounds,compositions, and methods for making and using the same, withoutdeparting from the scope of the invention herein, and it is intendedthat all matter contained in the above description shall be interpretedin an illustrative and not in a limiting sense.

We claim:
 1. A nanoparticle comprising: (i) magnetic core having one ormore magnetic metal oxide crystals; and (ii) a noncrosslinked polymercoating associated with the core, wherein the polymer coating has aplurality of carboxyl groups and plurality of reactive primary aminogroups, wherein a portion of the carboxyl groups are associated with thecrystals.
 2. The nanoparticle of claim 1 wherein reactive primary aminogroups are associated with polymer through a peptidyl linkage of theformula: —O—(CH₂)_(m)—CONH—[X]wherein X is —(CH₂)_(n) NH₂, —(CH₂)_(o)CHNHCOO—, —(CH₂)₃NH (CH₂)₄NH₂ or —(CH₂)₃NH(CH₂)₄NH(CH₂)₃ NH₂— whereinm=1,2, or 3; n=2,3, 6, and o=3 or
 4. 3. The nanoparticle of claim 1,wherein the magnetic core comprises one or more superparamagnetic ironoxide crystals.
 4. The nanoparticle of claim 3 wherein thesuperparamagnetic core has a diameter between about 1 nm and about 25nm.
 5. The nanoparticle of claim 4 where in the superparamagnetic corehas a diameter between about 3 nm and about 10 nm.
 6. The nanoparticleof claim 5 wherein the core has a diameter about 5 nm.
 7. Thenanoparticle of claim 1 wherein the nanoparticle has diameter betweenabout 15 nm and 100 nm.
 8. The nanoparticle of claim 7 wherein thenanoparticle has a diameter between about 20 nm and about 100 nm.
 9. Thenanoparticle of claim 1 wherein the coating is selected from the groupof polymers consisting of natural polymers, synthetic polymers andderivatives thereof.
 10. The nanoparticle of claim 1 wherein the polymeris carboxymethyldextran.
 11. The nanoparticle of claim 1 wherein thenanoparticle is conjugated with a biomolecule.
 12. The nanoparticle ofclaim 11 wherein the biomolecule has at least one sulfhydryl groupwherein the sulfhydryl group reacts with an amino group on the coating.13. A nanoparticle-bioconjugate molecule comprising: (i) a nanoparticle,the nanoparticle comprising (a) a magnetic core having one or moremagnetic metal oxide crystals; (b) a noncrosslinked polymer coatingassociated with the core, wherein the polymer coating has a plurality ofcarboxyl groups and plurality of reactive primary amino groups, whereina portion of the carboxyl groups are associated with the crystals; (ii)a biomolecule linked to the nanoparticle, wherein the biomolecule has atleast one sufhydryl group linked to the amino group.
 14. The ananoparticle-bioconjugate of claim 13 wherein the amino groups areassociated with polymer through a peptidyl linkage of the formula:—O—(CH₂)_(m)—CONH—[X]wherein X is —(CH₂)_(n) NH₂, —(CH₂)_(o)CH NHCOO—,—(CH₂)₃NH (CH₂)₄NH₂ or —(CH₂)₃NH(CH₂)₄NH(CH₂)₃ NH₂ wherein m=1, 2, or 3;n=2, 3, 6, and o=3 or
 4. 15. A process for synthesizing an aminefunctionalized magnetic metal oxide nanoparticle comprising: i.obtaining a polymer having a plurality of carboxyl groups attachedthereto, ii. contacting the polymer with magnetic metal oxide to producea coated magnetic metal oxide wherein a portion of the carboxyl groupsare associated with the metal oxide; iii. reacting the coated magneticmetal oxide of step (ii) with a diamine.
 16. The process of claim 15wherein the diamine is ethylene diamine.
 17. The process of claim 15wherein the diamine is hexane diamine.
 18. The process of claim 15wherein step (ii) further comprises the steps of (a) providing asolution of soluble iron salts; (b) converting the iron salts into ironoxide crystals; and (c) removing unassociated polymer.
 19. The processof claim 18 wherein the step of converting the iron salts into ironoxides further includes the steps of: (d) heating the iron salts to atemperature between about 4° C. and about 20° C.; (e) adding an amountof base to raise the pH to about 8 or higher to form iron oxides. 20.The process of claim 19 further including the step of heating the ironoxides to a temperature about 60° C. or more for at least 30 minutes.21. A pharmaceutical composition comprising: (A) Ananoparticle-bioconjugate molecule comprising: (i) a nanoparticle, thenanoparticle comprising (a) a magnetic core having one or more magneticmetal oxide crystals; (b) a noncrosslinked polymer coating associatedwith the core, wherein the polymer coating has a plurality of carboxylgroups and plurality of reactive primary amino groups, wherein a portionof the carboxyl groups are associated with the crystals; (ii) abiomolecule linked to the nanoparticle, wherein the biomolecule has atleast one sufhydryl group linked to at least one amino group; and (B) apharmaceutically acceptable carrier.